T Lymphocytes from Chagasic Patients Are Activated but Lack Proliferative Capacity and Down-Regulate CD28 and CD3ζ

Background

Chronic persistent infections have been associated with T lymphocytes functional impairment. The aim of this study was to compare the activation status, the proliferative potential and the expression of CD28 and CD3ζ chain on T lymphocytes between chronic chagasic patients and uninfected controls.

Methodology/Principal Findings

Forty-two chronic chagasic patients, 28 healthy individuals and 32 non-chagasic cardiomyopathy donors were included. Peripheral blood was marked for CD3, CD4, CD8, HLA-DR, CD28, CD38 and intracellular CD3ζ. Peripheral blood mononuclear cells were stained with carboxyfluorescein diacetate succinimidylester and incubated with T. cruzi lysate or phytohemagglutinin for five days. Cells from 3 healthy controls were incubated with T. cruzi trypomastigotes separated with transwells; and the expression of CD3ζ chain and proliferation index was determined. Heart-infiltrating cells from two chronic chagasic patients were tested for the aforementioned cellular markers. Chagasic patients displayed higher frequencies of CD4+/HLA-DR+/CD38+ (8.1%±6.1) and CD8+/HLA-DR+/CD38+ (19.8±8.9) T cells in comparison with healthy (1.6±1.0; 10.6±8.0) and non-chagasic cardiomyopathy donors (2.9±2.9; 5.8±6.8). Furthermore, the percentage of CD4+ activated T cells was higher in chagasic patients with cardiac involvement. CD8+ T cells proliferation index in chagasic donors (1.7±0.3) was lower when compared with healthy (2.3±0.3) and non-chagasic cardiomyopathy individuals (3.1±1.1). The frequencies of CD4+/CD28+ and CD8+/CD28+ T cells, as well as the CD3ζ^bright/CD3ζ^dim% ratios in CD4+ and CD8+ were lower in chagasic patients when compared with both control groups. The CD3ζ^bright/CD3ζ^dim% ratio and proliferative indexes for CD4+ and CD8+ T lymphocytes decreased gradually in those cells cultivated with parasites and displayed lower values than those incubated with medium alone. Finally, heart-infiltrating T cells from two T. cruzi infected patients also expressed activation markers and down-regulate CD28 and CD3ζ.

Conclusions

CD8+ T lymphocytes from chagasic donors displayed reduced proliferative capacity, which might be associated with CD3ζ down-regulation and diminished CD28 expression on CD4 T cells.     

Genome-wide methylation profiling reveals Zinc finger protein 516 (ZNF516) and FK-506-binding protein 6 (FKBP6) promoters frequently methylated in cervical neoplasia,associated with HPV status and ethnicity in a Chilean population

Cervical cancer is a major health concern among women in Latin America due to its high incidence and mortality. Therefore, the discovery of molecular markers for cervical cancer screening and triage is imperative. The aim of this study was to use a genome wide DNA methylation approach to identify novel methylation biomarkers in cervical cancer. DNA from normal cervical mucosa and cervical cancer tissue samples from Chile was enriched with Methylated DNA Immunoprecipitation (MeDIP), hybridized to oligonucleotide methylation microarrays and analyzed with a stringent bioinformatics pipeline to identify differentially methylated regions (DMRs) as candidate biomarkers. Quantitative Methylation Specific PCR (qMSP) was used to study promoter methylation of candidate DMRs in clinical samples from two independent cohorts. HPV detection and genotyping were performed by Reverse Line Blot analysis. Bioinformatics analysis revealed GGTLA4, FKBP6, ZNF516, SAP130, and INTS1 to be differentially methylated in cancer and normal tissues in the Discovery cohort. In the Validation cohort FKBP6 promoter methylation had 73% sensitivity and 80% specificity (AUC = 0.80). ZNF516 promoter methylation was the best biomarker, with both sensitivity and specificity of 90% (AUC = 0.92), results subsequently corroborated in a Prevalence cohort. Together, ZNF516 and FKBP6 exhibited a sensitivity of 84% and specificity of 81%, when considering both cohorts. Our genome wide DNA methylation assessment approach (MeDIP-chip) successfully identified novel biomarkers that differentiate between cervical cancer and normal samples, after adjusting for age and HPV status. These biomarkers need to be further explored in case-control and prospective cohorts to validate them as cervical cancer biomarkers.     

Upregulation of ERK1/2-eNOS via AT2 Receptors Decreases the Contractile Response to Angiotensin II in Resistance Mesenteric Arteries from Obese Rats

It has been clearly established that mitogen-activated protein kinases (MAPKS) are important mediators of angiotensin II (Ang II) signaling via AT1 receptors in the vasculature. However, evidence for a role of these kinases in changes of Ang II-induced vasoconstriction in obesity is still lacking. Here we sought to determine whether vascular MAPKs are differentially activated by Ang II in obese animals. The role of AT2 receptors was also evaluated. Male monosodium glutamate-induced obese (obese) and non-obese Wistar rats (control) were used. The circulating concentrations of Ang I and Ang II, determined by HPLC, were increased in obese rats. Ang II-induced isometric contraction was decreased in endothelium-intact resistance mesenteric arteries from obese compared with control rats and exhibited a retarded AT1 receptor antagonist response. Blocking of AT2 receptors and inhibition of either endothelial nitric oxide synthase (eNOS) or extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) restored Ang II-induced contraction in obese rats. Western blot analysis revealed increased protein expression of AT2 receptors in arteries from obese rats. Basal and Ang II-induced ERK1/2 phosphorylation was also increased in obese rats. Blockade of either AT1 or AT2 receptors corrected the increased ERK1/2 phosphorylation in arteries from obese rats to levels observed in control preparations. Phosphorylation of eNOS was increased in obese rats. Incubation with the ERK1/2 inhibitor before Ang II stimulation did not affect eNOS phosphorylation in control rats; however, it corrected the increased phosphorylation of eNOS in obese rats. These results clearly demonstrate that enhanced AT2 receptor and ERK1/2-induced, NO-mediated vasodilation reduces Ang II-induced contraction in an endothelium-dependent manner in obese rats.     

Expression of miR-15/107 Family MicroRNAs in Human Tissues and Cultured Rat Brain Cells

The miR-15/107 family comprises a group of 10 paralogous microRNAs （miRNAs）,sharing a 5＇ AGCAGC sequence.These miRNAs have overlapping targets.In order to characterize the expression of miR-15/107 family miRNAs,we employed customized TaqMan Low-Density micro-fluid PCR-array to investigate the expression of miR-15/107 family members,and other selected miRNAs,in 11 human tissues obtained at autopsy including the cerebral cortex,frontal cortex,primary visual cortex,thalamus,heart,lung,liver,kidney,spleen,stomach and skeletal muscle.miR-103,miR-195 and miR-497 were expressed at similar levels across various tissues,whereas miR-107 is enriched in brain samples.We also examined the expression patterns of evolutionarily conserved miR-15/107 miRNAs in three distinct primary rat brain cell preparations （enriched for cortical neurons,astrocytes and microglia,respectively）.In primary cultures of rat brain cells,several members of the miR-15/107 family are enriched in neurons compared to other cell types in the central nervous system （CNS）.In addition to mature miRNAs,we also examined the expression of precursors （pri-miRNAs）.Our data suggested a generally poor correlation between the expression of mature miRNAs and their precursors.In summary,we provide a detailed study of the tissue and cell type-specific expression profile of this highly expressed and phylogenetically conserved family of miRNA genes.     

Follow-up of an Asymptomatic Chagas Disease Population of Children after Treatment with Nifurtimox (Lampit) in a Sylvatic Endemic Transmission Area of Colombia

Background

Chagas disease is an anthropozoonosis caused by Trypanosoma cruzi. Two drugs are currently used for the etiological treatment of the disease: Nifurtimox (Lampit) and Benznidazole. This study presents a quasi-experimental trial (non-control group) of sixty-two patients who were treated for Chagas disease with Nifurtimox (Lampit), and were then followed for 30 months post-treatment. The safety of Nifurtimox (Lampit) for Chagas disease in this group of children primarily between 4 and 19 years old was also evaluated.

Materials and methods

The 62 patients included in the study were selected when resulted seropositive for two out of three fundamentally different serological tests. All children were treated during two months according to protocols established by WHO. Monitoring was performed every twenty days to evaluate treatment safety. In 43 patients, two different serological tests: ELISA and IFAT; and two parasitological tests: blood culture, and real time PCR, (qPCR) were performed to assess therapeutic response, defined as post-treatment serological negativization.

Principal findings

All patients completed the treatment successfully, and six patients abandoned the post-treatment follow-up. Adverse effects occurred in 74% of patients, but only 4.8% of cases required temporary suspension to achieve 100% adherence to the 60-day treatment, and all symptoms reverted after treatment completion. Both parasite load (measured through qPCR) and antibodies (ELISA absorbance) evidenced a significant median reduction 6 months after treatment from 6.2 to 0.2 parasite equivalents/mL, and from 0.6 to 0.2 absorbance units respectively (p<0.001). Serological negativization by ELISA was evident since 6 months post-treatment, whereas by IFAT only after 18 months. Serological negativization by the two tests (ELISA and IFAT) was 41.9% (95%CI: 26.5–57.3) after 30 months post-treatment. qPCR was positive in 88.3% of patients pre-treatment and only in 12.1% of patients after 30 months. Survival analysis indicated that only 26.3% (95%CI: 15.5–44.8) persisted with negative qPCR during the whole follow-up period.

Conclusions

Nifurtimox was very well tolerated and successfully reduced parasite load and antibody titers. Re-infection, lysed parasites or a lack of anti-parasitic activity could explain these persistently positive qPCR cases.     

A Randomized Blinded Study of the Left Ventricular Myocardial Performance Index Comparing Epinephrine to Levosimendan following Cardiopulmonary Bypass

Background

The objective was to evaluate the effect of epinephrine and levosimendan on the left ventricle myocardial performance index in patients undergoing on-pump coronary artery by-pass grafting (CABG).

Methods

In a double-blind, randomized clinical trial, 81 patients (age: 45–65 years) of both genders were randomly divided to receive either epinephrine at a dosage of 0.06 mcg.kg¹.min^-1 (epinephrine group, 39 patients) or levosimendan at 0.2 mcg.kg¹.min^-1 (levosimendan group, 42 patients) during the rewarming of cardiopulmonary by-pass (CPB). Hemodynamic data were collected 30 minutes after tracheal intubation, before chest open (pre-CPB) and 10 minutes after termination of protamine (post-CPB). As the primary outcome, we evaluated the left ventricle myocardial performance index by the Doppler echocardiography. The myocardial performance index is the sum of the isovolumetric contraction time and the isovolumetric relaxation time, divided by the ejection time. Secondary outcomes were systolic and diastolic evaluations of the left ventricle and postoperative troponin I and MB-CK levels.

Results

Of the 81 patients allocated to the research, we excluded 2 patients in the epinephrine group and 6 patients in the levosimendan group because they didn’t wean from CPB in the first attempt. There was no statistical difference between the groups in terms of patient characteristics, risk factors, or CPB time. The epinephrine group had a lower left ventricle myocardial performance index (p = 0.0013), higher cardiac index (p = 0.03), lower systemic vascular resistance index (p = 0.01), and higher heart rate (p = 0.04) than the levosimendan group at the post-CPB period. There were no differences between the groups in diastolic dysfunction. The epinephrine group showed higher incidence of weaning from CPB in the first attempt (95% vs 85%, p = 0.0001) when compared to the levosimendan group and the norepinephrine requirement was higher in the levosimenandan group than epinephrine group (16% vs. 47%; p = 0.005) in post-CPB period. Twenty-four hours after surgery, the plasma levels of troponin I (epinephrine group: 4.5 ± 5.7 vs. levosimendan group: 2.5 ± 3.2 g/dl; p = 0.09) and MB-CK (epinephrine group: 50.7 ± 31 vs. levosimendan group: 37 ± 17.6 g/dl; p = 0.08) were not significantly different between the two groups.

Conclusion

When compared to levosimendan, patients treated with epinephrine had a lower left ventricle myocardial performance index in the immediate post-CPB period, encouraging an efficient weaning from CPB in patients undergoing on-pump CABG.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01616069","term_id":"NCT01616069"}}NCT01616069     

Shared Genetic Factors of Anxiety and Depression Symptoms in a Brazilian Family-Based Cohort,the Baependi Heart Study

To investigate the phenotypic and genetic overlap between anxiety and depression symptoms in an admixed population from extended family pedigrees. Participants (n = 1,375) were recruited from a cohort of 93 families (mean age±SD 42±16.3, 57% female) in the rural town of Baependi, Brazil. The Hospital Anxiety and Depression Scale (HADS) was used to assess depression and anxiety symptoms. Heritability estimates were obtained by an adjusted variance component model. Bivariate analyses were performed to obtain the partition of the covariance of anxiety and depression into genetic and environmental components, and to calculate the genetic contribution modulating both sets of symptoms. Anxiety and depression scores were 7.49±4.01 and 5.70±3.82, respectively. Mean scores were affected by age and were significantly higher in women. Heritability for depression and anxiety, corrected for age and sex, were 0.30 and 0.32, respectively. Significant genetic correlations (ρ_g = 0.81) were found between anxiety and depression scores; thus, nearly 66% of the total genetic variance in one set of symptoms was shared with the other set. Our results provided strong evidence for a genetic overlap between anxiety and depression symptoms, which has relevance for our understanding of the biological basis of these constructs and could be exploited in genome-wide association studies.